Geography-linked phylogeny of theDamaster ground beetles inferred from mitochondrial ND5 gene sequences

We have constructed phylogenetic trees based on sequence comparisons of mitochondrial NADH dehydrogenase subunit 5 gene for 11Damaster blaptoides specimens from various localities of Japan. The specimens consist of eight subspecies.Coptolabrus fruhstorferi, Acoptolabrus gehinii, andProcrustes kolbei, which are taxonomically related toDamaster, have also been analyzed for comparison.Damaster is more related toAcoptolabrus than toCoptolabrus in the ND5 trees, in contrast to the generally accepted view thatDamaster is derived fromCoptolabrus, whereasAcoptolabrus is the sister group ofDamaster/Coptolabrus. The emergence ofProcrustes is much earlier than the rest of the genera. TheDamaster subspecies are monophyletic. Four major lineages are recognized, which are geography linked within the Japanese archipelago. The origin and diversification process have been discussed based on these findings.The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with the accession numbers D50422-D50429     

Parallel evolution in radiation ofOhomopterus ground beetles inferred from mitochondrial ND5 gene sequences

Molecular phylogenetic analyses using mitochondrial NADH dehydrogenase subunit 5 (ND5) gene sequences representing all 15 species and the majority of subspecies or races of theOhomopterus ground beetles from all over the Japanese archipelago have uncovered a remarkable evolutionary history. Clustering of the species in the molecular phylogenetic tree is linked to their geographic distribution and does not correlate with morphological characters. Taxonomically the same species or the members belonging to the same species-group fall out in more than two different places on the ND5 tree. Evidence has been presented against a possible participation of ancestral polymorphism and random lineage sorting or of hybrid individuals for the observed distribution of mitochondrial DNA haplotypes. The most plausible explanation of our results is that parallel evolution took place in different lineages. Most notably,O. dehaanii, O. yaconinus, andO. japonicus in a lineage reveal almost identical morphology with those of the same species (or subspecies) but belonging to the phylogenetically remote lineages.The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with accession numbers D50711-DD-50733 and D87131-D87186.     

Phylogenetic placement of the basidiomycetous yeastsKondoa malvinella andRhodosporidium dacryoidum,and the anamorphic yeastSympodiomycopsis paphiopedili by means of 18S rRNA gene sequence analysis

The 18S ribosomal RNA gene sequences of the basidiomycetous yeastsKondoa malvinella andRhodosporidium dacryoidum, and an anamorphic yeastSympodiomycopsis paphiopedili were determined. The 18S rRNA gene ofR. dacryodium IAM 13522 (ex type) revealed the presence of an intron-like region with a length of 404 nucleotides, which is presumably assigned to a group I intron. The phylogenetic tree, including 34 published reference sequences, was inferred from 1493 sites which could be unambiguously aligned. The molecular phylogeny, using the ascomycetes as an outgroup, divided the basidiomycetes into three major lineages. The first lineage was composed of the smut fungi (Ustilaginales), represented byUstilago maydis, U. hordei, andTilletia caries, includingS. paphiopedili. The second lineage included the type species of teliospore-forming yeast generaLeucosporidium, Rhodosporidium, andSporidiobolus, and the generaErythrobasidium andKondoa, both previously included in the Filobasidiaceae.Rhodosporidium dacryodium showed a close relationship withE. hasegawianum, which was backed by a high bootstrap support. The rust fungiCronartium ribicola andPeridermium harknessii were also included in this lineage. The last lineage was formed by the filobasidiaceous yeasts,Cystofilobasidium capitatum, Mrakia frigida, Filobasidium floriforme, andFilobasidiella neoformans, and the anamorphic yeastsBullera alba (the anamorph ofBulleromyces albus) andTrichosporon cutaneum. Members ofTremella and selected hymenomycetous genera were also included in this lineage.The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the following accession numbers D13459 (Rhodosporidium dacryodium), D13776 (Kondoa malvinella), and D14006 (Sympodiomycopsis paphiopedili).     

Systematics, biogeography and diversification of the Indo-Australian genus Delias Hübner (Lepidoptera: Pieridae): phylogenetic evidence supports an 'out-of-Australia' origin

Abstract Two alternative hypotheses for the origin of butterflies in the Australian Region, that elements dispersed relatively recently from the Oriental Region into Australia (northern dispersal hypothesis) or descended from ancient stocks in Gondwana (southern vicariance hypothesis), were tested using methods of cladistic vicariance biogeography for the Delias group, a diverse and widespread clade in the Indo‐Australian Region. A phylogenetic hypothesis of the twenty‐four species‐groups recognized currently in Delias and its sister genus Leuciacria is inferred from molecular characters generated from the nuclear gene elongation factor‐1 alpha (EF‐1α) and the mitochondrial genes cytochrome oxidase subunits I and II (COI/COII) and NADH dehydrogenase 5 (ND5). Phylogenetic analyses based on maximum parsimony, maximum likelihood and Bayesian inference of the combined dataset (3888 bp, 1014 parsimony informative characters) confirmed the monophyly of Delias and recovered eight major lineages within the genus, informally designated the singhapura, belladonna, hyparete, chrysomelaena, eichhorni, cuningputi, belisama and nigrina clades. Species‐group relationships within these clades are, in general, concordant with current systematic arrangements based on morphology. The major discrepancies concern the placement of the aganippe, belisama and chrysomelaena groups, as well as several species‐groups endemic to mainland New Guinea. Two species (D. harpalyce (Donovan), D. messalina Arora) of uncertain group status are currently misplaced based on strong evidence of paraphyly, and are accordingly transferred to the nigrina and kummeri groups, respectively. Based on this phylogeny, a revised systematic classification is presented at the species‐group level. An historical biogeographical analysis of the Delias group revealed that the most parsimonious reconstruction is an origin in the Australian Region, with at least seven dispersal events across Wallacea to the Oriental Region. The eight major clades of Delias appear to have diverged rapidly following complete separation of the Australian plate from Gondwana and its collision with the Asian plate in the late Oligocene. Further diversification and dispersal of Delias in the Miocene–Pliocene are associated with major geological and climatic changes that occurred in Australia–New Guinea during the late Tertiary. The ‘out‐of‐Australia’ hypothesis for the Delias group supports an origin of the Aporiina in southern Gondwana (southern vicariance hypothesis), which proposes that the ancestor of Delias + Leuciacria differentiated vicariantly on the Australian plate.     

Discrepancy in divergence of the mitochondrial and nuclear genomes ofDrosophila teissieri andDrosophila yakuba

Summary Restriction sites were compared in the mitochondrial DNA (mtDNA) molecules from representatives of two closely related species of fruit flies: nine strains ofDrosophila teissieri and eight strains ofDrosophila yakuba. Nucleotide diversities amongD. teissieri strains and amongD. yakuba strains were 0.07% and 0.03%, respectively, and the nucleotide distance between the species was 0.22%. Also determined was the nucleotide sequence of a 2305-nucleotide pari (ntp) segment of the mtDNA molecule ofD. teissieri that contains the noncoding adenine+thymine (A+T)-rich region (1091 ntp) as well as the genes for the mitochondrial small-subunit rRNA, tRNA^f-met, tRNA^gln, and tRNA^ile, and portions of the ND2 and tRNA^val genes. This sequence differs from the corresponding segment of theD. yakuba mtDNA by base substitutions at 0.1% and 0.8% of the positions in the coding and noncoding regions, respectively. The higher divergence due to base substitutions in the A+T-rich region is accompanied by a greater number of insertions/deletions than in the coding regions. From alignment of theD. teissieri A+T-rich sequence with those ofD. yakuba andDrosophila virilis, it appears that the 40% of this sequence that lies adjacent to the tRNA^ile gene has been highly conserved. Divergence between the entireD. teissieri andD. yakuba mtDNA molecules, estimated from the sequences, was 0.3%; this value is close to the value (0.22%) obtained from the restriction analysis, but 10 times lower than the value estimated from published DNA hybridization results. From consideration of the relationships of mitochondrial nucleotide distance and allozyme genetic distance found among seven species of theDrosophila melanogaster subgroup, the mitochondrial nucleotide distance observed forD. teissieri andD. yakuba is anomalously low in relation to the nuclear genetic distance.     

Molecular reexamination of korean umbelliferae based on internal transcribed spacer sequences of rDNA:Ligusticum tenuissimum (Nakai) Kitagawa andLibanotis coreana (Wolff) kitagawa

The taxonomic status ofLigusticum tenuissimum (Nakai) Kitagawa andLibanotis coreana (Wolff) Kitagawa has been controversial in Korea. We examined their phylogenetic relationships by comparing the internal transcribed spacer (=ITS) sequences of the nuclear ribosomal RNA gene (=rDNA) with another seven species in the Korean Umbelliferae. Our results support the legitimacy of the nomenclatural combination withL. tenuissimum Kitagawa rather than withAngelica tenuissima Nakai. We also suggest usingL coreana (Wolff) Kitagawa instead ofSeseli coreanum Wolff becauseL. coreana has been completely separated from most taxa of the genusSeseli. In addition, we have confirmed at least three phylogenetic lines in the genusLigusticum. One is the core group ofLigusticum that comprisesL tenuissimum and four other species. This clade includes theLigusticum hultenii line andLigusticum scothicum, known as an anomaly ofLigusticum. The second clade is composed ofLigusticum physospermifolium withSeseli monatum, which indicates the polyphyly ofLigusticum. The third line isLigusticum acutilobum, clustered as a sister grade ofDystaenia.     

Serologische Untersuchungen zur Gliederung derBrassicaceae

The serological investigations support the opinion ofJanchen (1942) to combine the generaBunias, Isatis, andSisymbrium in the tribeSisymbrieae; Cheiranthus, Erysimum, andMatthiola in the tribeHesperideae; andBrassica, Crambe, Sinapis, andSuccowia in the tribeBrassiceae. They further underline the central position of theSisymbrieae and the isolated position of theHeliophileae. In accordance withEigner (1973) theBrassiceae are placed closer to theSisymbrieae than inJanchen; the same holds for thePringleeae. No serological justification could be found to uniteArabis andBarbarea in the tribeArabideae, andAlyssum andLunaria in theAlysseae. From the antigen-systems used among the representatives ofJanchen's Lepidieae the generaLepidium andNeslia show remarkable correspondence both toCamelina andThlaspi, but not toCochlearia which appears distant fromCamelina andThlaspi also.

Teil 1/Part 1.     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Drosophila mitochondrial DNA: Conserved sequences in the A+T-rich region and supporting evidence for a secondary structure model of the small ribosomal RNA

Summary The sequence of a segment of theDrosophila virilis mitochondrial DNA (mtDNA) molecule that contains the A+T-rich region, the small rRNA gene, the tRNA^f-met, tRNA^gln, and tRNA^ile genes, and portions of the ND2 and tRNA^val genes is presented and compared with the corresponding segment of theD. yakuba mtDNA molecule. The A+T-rich regions ofD. virilis andD. yakuba contain two correspondingly located sequences of 49 and 276/274 nucleotides that appear to have been conserved during evolution. In each species the replication origin of the mtDNA molecule is calculated to lie within a region that overlaps the larger conserved sequence, and within this overlap is found a potential hairpin structure. Substitutions between the larger conserved sequences of the A+T-rich regions, the small mt-rRNA genes, and the ND2 genes are biased in favor of transversions, 71–97% of which are AT changes. There is a 13.8 times higher frequency of nucleotide differences between the 5 halves than between the 3 halves of theD. virilis andD. yakuba small mt-rRNA genes. Considerations of the effects of observed substitutions and deletion/insertions on possible nucleotide pairing within the small mt-rRNA genes ofD. virilis andD. yakuba strongly support the secondary structure model for theDrosophila small mt-rRNA that we previously proposed.     

Molecular phylogeny of the genus<Emphasis Type="Italic">Hypericum</Emphasis> (Hypericaceae) from Korea and Japan: evidence from nuclear rDNA ITS sequence data

As part of our ongoing phylogenetic study of genusHypericum, nuclear ribosomal DNA internal transcribed spacer sequences were analyzed for 36 species ofHypericum as ingroup and two species ofThornea as outgroup. This sampling included most of the previously described species from both Korea and Japan. The ITS phylogeny suggested that the surveyedHypericum species belong to a monophyletic section,Trigynobrathys, and a polyphyletic section,Hypericum. In addition, two monotypic sections,Sampsonia andRoscyna, were identified. Members of sectionHypericum occur in four different lineages worldwide, which imply at least four independent origins. The Korean and Japanese species of sectionHypericum form a monophyletic group, except forH. vulcanicum. Instead, that particular species belongs to a distinct monophyletic group withH. scoreri andH. formosa from other geographic areas, and is a sister to sectionTrigynobrathys. The Korean and Japanese species of sectionTrigynobrathys show a monophyletic origin.H. sampsonii is now recognized as a distinct section rather than being a member of sectionsHypericum orDrosocarpium, as had been indicated previously. Our results differ somewhat from those of recent morphological and cytological studies. The phylogenetic relationships among Korean and Japanese species have now been mostly resolved via ITS phylogeny.     

Radiation and reticulation: extensive introgressive hybridization in the carabid beetles Ohomopterus inferred from mitochondrial gene genealogy

 In the evolutionary process of an animal lineage, interactions in secondary contacts of differentiated populations and introgressive hybridization may play an important role. In the Japanese islands, the carabid subgenus Ohomopterus (genus Carabus) exhibits a marked differentiation in body size and genital morphology. Although geographical differentiation is apparent, two or three species usually coexist at many localities. Their reproductive isolation relies on body size differences, chemical cues for mate recognition, and a species-specific genital lock-and-key system. However, these isolation mechanisms are not always effective enough to prevent interspecific hybridization. An initial assessment of the species-level phylogeny with mitochondrial gene sequences revealed that the gene genealogy is highly inconsistent with the morphology-based taxonomy. A comparison of mitochondrial and nuclear gene genealogies showed that these are strongly incongruent with each other, while the nuclear gene genealogy is more consistent with traditional taxonomy, indicating the repeated occurrence of introgression of mitochondria across species. Here, two different cases of mitochondrial introgression among Ohomopterus species are described in detail, one for parapatric species and the other for sympatric species. First, mitochondrial haplotypes and sequences were studied in Carabus insulicola and three taxa parapatric with C. insulicola, at least two of which hybridize with C. insulicola naturally. Among the four species studied, directional introgressions of mitochondria across boundary zones were detected. Second, in the Mt. Kongo area in central Honshu, which harbors five species, introgression of mitochondria among four out of the five species was detected, despite the apparent absence of on-going natural hybridization. These inferred cases of mitochondrial introgression indicate that species interactions through hybridization could have played an important role at various stages in the evolution of Ohomopterus. Received: April 12, 2002 / Accepted: October 17, 2002 Acknowledgments I am grateful to Alfried P. Vogler for a long-lasting collaboration in the molecular phylogenetic study of Ohomopterus. R. Ishikawa, K. Kubota, M. Ujiie, Y. Takami, and F. Kusumoto have also collaborated at various stages of this study. Thanks are also due to K. Miyashita, T. Funakoshi, H. Fujimoto, T. Dejima, Y. Nagahata, T. Miyagawa, K. Yodoe, H. Kadowaki, S. Nakamine, Y. Oka, H. Tanaka, T. Tanabe, K. Kusakari, and T. Okumura for their care of specimens. Supported by grants-in-aid from the Japan Society for the Promotion of Science (Nos. 09640748, 11304056).     

Evolution of thedec-1 eggshell locus inDrosophila. I. Restriction site mapping and limited sequence comparison in themelanogaster species subgroup

Summary We have analyzed 18 kb of DNA in and upstream of thedefective chorion-1 (dec-1) locus of the eight known species of themelanogaster species subgroup ofDrosophila. The restriction maps ofD. simulans, D. mauritiana, D. sechellia, D. erecta, andD. orena are shown to have basically the restriction map ofD. melanogaster, whereas the maps ofD. teissieri andD. yakuba were more difficult to align. However, the basic amount of DNA and sequence arrangement appear to have been conserved in these species. A small deletion of varying length (65–200 bp) is found in a repeated sequence of the central transcribed region ofD. melanogaster, D. simulans, andD. erecta. Restriction site mapping indicated that thedec-1 gene is highly conserved in themelanogaster species subgroup. However, sequence comparison revealed that the amount of nucleotide and amino acid substitution in the repeated region is much larger than in the 5 translated region. The 5 flanking region showed noticeable restriction site polymorphisms between species. Based on calculations from the restriction maps a dendrogram was derived that supports earlier published phylogenetic relationships within themelanogaster species subgroup except that theerecta-orena pair is placed closer to themelanogaster complex than toD. teissieri andD. yakuba.     

Phylogenetic relationships of silver crusian carp Carassius auratus gibelio, C. auratus cuvieri, crucian carp Carassius carassius, and common carp Cyprinus carpio as inferred from mitochondrial DNA variation

PCR-RFLP analysis of the ND3/ND4L/ND4 and 12S/16S rRNA regions and nucleotide sequence variation of the cytochrome b gene were used to study the mtDNA divergence in species of the family Cyprinidae, to examine the phylogenetic relationships of the species, and to identify their taxonomic status. The results indicated that an ancestral form diverged into silver crucian carp and crucian carp after its separation from the common carp lineage. The divergence of continental Carassius auratus gibelio and Japanese C. auratus cuvieri occurred more recently. Two well distinguishable mtDNA phylogroups, suggesting divergent evolution, were observed in continental C. auratus gibelio populations. The divergence was possibly related to the formation of two silver crucian carp groups with different types of reproduction, triploid gynogenetic and diploid gonochoric. At the same time, the results supported the high probability of current genetic exchange between the forms. In view of these findings and high morphological similarity of the two forms, they were not considered to be separate species.     

Phylogenetic relationships among the skunks: A molecular perspective

Phylogenetic relationships among the three recognized genera of skunks (Mephitis, Spilogale, andConepatus) were examined using allozymes and nucleotide sequence data from portions of the cytochromeb gene and displacement loop of the mitochondrial genome. The data sets indicated thatMephitis andSpilogale shared a common ancestor after they diverged fromConepatus. Intrageneric relationships revealed that the level of genetic divergence between the two species ofSpilogale was equal to the level of divergence between the two species ofMephitis, with the two North American species ofConepatus possessing the least amount of genetic divergence.     

The branching of the inflorescence and vegetative shoot and taxonomy of the genusKummerowia (Leguminosae)

A study of the branching of the inflorescence and the vegetative shoot of the genusKummerowia, consisting ofK. stipulacea (Maxim.) Makino andK. striata (Thunb.) Schindler, has led to the following conclusions: (1) the inflorescences of both species are reduced compound cymes, (2) the branching system of the inflorescence ofKummerowia is not clearly different from that of the vegetative shoot and there are some transitional forms between both systems, and (3) the inflorescence ofKummerowia is different from the racemose inflorescences ofLespedeza andCampylotropis. Based on the differences found in the branching system of the inflorescence,Kummerowia is distinctly separated fromLespedeza andCampylotropis and is more correctly treated as a distinct genus from the latter two.     

Evolutionary relationship ofHLA-DRB genes inferred from intron sequences

The major histocompatibility complex (Mhc) consists of class I and class II genes. In the humanMhc (HLA) class II genes, nineDRB loci have been identified. To elucidate the origin of these duplicated loci and allelic divergences at the most polymorphicDRBI locus, introns 4 and 5 as well as the 3′ untranslated region (altogether approximately 1,000 base pairs) of sevenHLA-DRB loci, threeHLA-DRBI alleles, and nine nonhuman primateDRB genes were examined. It is shown that there were two major diversification events inHLA-DRB genes, each involving gene duplications and allelic divergences. Approximately 50 million years (my) ago,DRBI ^*04 and an ancestor of theDRB1 ^*03 cluster (DRBI ^*03, DRBI^*15, andDRB3) diverged from each other andDRB5, DRB7, DRB8, and an ancestor of theDRB2 cluster (DRB2, DRB4, andDRB6) arose by gene duplication. Later, about 25 my ago,DRBI ^*15 diverged fromDRBI*03, andDRB3 was duplicated fromDRBI ^*03. Then, some 20 my ago, the lineage leading to theDRB2 cluster produced two new loci,DRB4 andDRB6. TheDRBI ^*03 andDRBI ^*04 allelic lineages are extraordinarily old and have persisted longer than some duplicated genes. The orthologous relationships ofDRB genes between human and Old World monkeys are apparent, but those between Catarrhini and New World monkeys are equivocal because of a rather rapid expansion and contraction of primateDRB genes by duplication and deletion. Correspondence to: Y. Satta     

Molecular evidence for the phylogenetic position ofHanabusaya asiatica Nakai (Campanulaceae), an endemic species in Korea

The phylogenetic relationship of the Korean endemic genus,Hanabusaya, to other campanulaceous genera has been controversial since it was described by Nakai in 1911. Three genera of Campanuloideae,Symphyandra, Adenophora, andCampanula, have been considered closely related by various taxonomists on the basis of anther shape, gross morphology, and pollen characters, respectively. We have tested these competing taxonomic hypotheses using the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA from 12 taxa representing 7 genera of Campanulaceae. The molecular phylogeny indicates strongly thatHanabusaya is more closely allied toAdenophora than toCampanula orSymphyandra. The phylogenetic affinity ofHanabusaya andAdenophora is supported by a 100% bootstrap value and a high decay index (13). The average sequence divergence value (Kimura’s 2-parameter method) betweenHanabusaya and theAdenophora species is 2.58. The value is significantly (about ten times) lower than the ones observed betweenHanabusaya and the species ofCampanula (average of 23.52) and betweenHanabusaya andSymphyandra (24.95). The ITS sequence phylogeny suggests that some morphological characters, such as fused anthers and corolla shape, are homoplastic in the Campanulaceae genera.     

Evolution of flower shape inVeroniceae (Scrophulariaceae)

Floral evolution in the tribeVeroniceae was examined using phylogenetic analysis combining 24 adult morphology and chromosome number characters with 22 qualitative and quantitative floral development characters. Taxa sampled included nine species ofVeroniceae and as an outgroup one species each ofDigitaleae andVerbasceae. Veronica, Besseya, andSynthyris formed one clade, subtended byPseudolysimachion and then by theHebe group;Veronicastrum orWulfenia represent the basal-most branch of the tribe. The ancestral flowers of theVeroniceae may have been small with moderately short corolla tubes and lobes; long corolla tubes arose four times in the tribe and large corolla lobes twice.     

Structure and expression of a nuclear gene for the PSI-D subunit of photosystem I inNicotiana sylvestris

The PSI-D subunit is the ferredoxin-binding site of photosystem I, and is encoded by the nuclear genepsaD. We isolated apsaD genomic clone fromNicotiana sylvestris, by screening a genomic library with apsaD cDNA which we previously cloned fromN. sylvestris (Yamamotoet al., Plant Mol Biol 17: 1251, 1991). Nucleotide sequence analysis revealed that this genomic clone contains apsaD gene, which does not correspond to thepsaD cDNA, so we designated these genespsaDb andpsaDa, respectively. ThepsaDb clone encodes a protein of 214 amino acids uninterrupted by introns. The N-terminal sequence determined for theN. sylvestris PSI-D protein encoded bypsaDb begins at the 49th residue. The products ofpsaDa andpsaDb share 82.7% and 79.5% identity at the amino acid and nucleotide levels, respectively. Genomic Southern analysis showed that two copies ofpsaD are present in theN. sylvestris genome. Ribonuclease protection assays and immunoblot analysis inN. sylvestris indicate that both genes are expressed in leaves, stems and flower buds, but neither is expressed in roots. During leaf development, the ratio ofpsaDb topsaDa mRNA increases from 0.12 in leaf buds to 0.36 in mature leaves. The relative abundance of the corresponding proteins decreased over the same developmental period. These results indicate that differential regulation mechanisms controlpsaDa andpsaDb expression at both the mRNA and protein levels during leaf development.